Lynch syndrome (LS) is a hereditary disease that predisposes patients to colorectal, endometrial, ovarian and other cancers. Definitive diagnosis of LS depends on detection of a deleterious, germline mutation in one of the DNA mismatch repair (MMR) genes. A problem arises for clinicians and genetic counselors, however, when the sequencing reveals a variation, such as a missense mutation, whose effect on gene function is not immediately obvious. These variants of uncertain significance (VUS) cannot be used to confirm nor rule out LS resulting in uncertainty about how to manage the patient and their family members. Laboratory studies to determine whether a VUS disrupts protein function has become an important strategy for determining their relevance to disease. Studying the function of a VUS in human cells is the most thorough approach to determining its effects on MMR function, however, this requires development of the proper cell culture model. We hypothesize that human pluripotent stem cells (PSC) can serve as an ideal model system for testing the functional effects of MMR gene variants. PSCs are immortal, which makes them a practical model system in the laboratory, yet non-transformed, thus they may more closely resemble the environment in which the variant protein first influences early transformation events in LS patient cells. In addition, they are pluripotent which means they can be differentiated into multiple cell types to examine cell-type specific effects. Our goal is to test whether PSCs can serve as an effective model system for testing the function of genetic variants by utilizing advanced techniques for genetic manipulation in these cells. The aims of this proposal include: 1) Examining the MMR pathway in various PSC lines and differentiated cell types derived from these cells. We will test for MMR protein expression, repair of G/T mismatches and ability to activate cell cycle checkpoints and/or apoptosis in response to different DNA damaging agents. 2) Examining whether likely cancer- associated variants of the MMR gene MSH2 disrupt cellular repair and response functions in PSCs. We will perform TALEN-mediated gene targeting at the endogenous MSH2 gene locus to create non-transformed cell culture models expressing different MSH2 VUS. These cells will be studied at different stages of differentiation including as intestinal epithelial-like cells to examine the effects of the VUS on DNA repair, cell cycle checkpoint and cell death signaling functions. This proposal will allow us to establish a new cell culture model system that can be used to help define the clinical significance of different MMR gene VUS, while also improving the overall understanding of the MMR pathway to benefit all patients suffering from MMR-defective cancers.